Arthritogenic alphaviruses: epidemiological and clinical perspective on emerging arboviruses.

Mosquito-borne viruses, or arboviruses, have been part of the infectious disease landscape for centuries, and are often, but not exclusively, endemic to equatorial and subtropical regions of the world. The past two decades saw the re-emergence of arthritogenic alphaviruses, a genus of arboviruses that includes several members that cause severe arthritic disease. Recent outbreaks further highlight the substantial public health burden caused by these viruses. Arthritogenic alphaviruses are often reported in the context of focused outbreaks in specific regions (eg, Caribbean, southeast Asia, and Indian Ocean) and cause debilitating acute disease that can extend to chronic manifestations for years after infection. These viruses are classified among several antigenic complexes, span a range of hosts and mosquito vectors, and can be distributed along specific geographical locations. In this Review, we highlight key features of alphaviruses that are known to cause arthritic disease in humans and outline the present findings pertaining to classification, immunogenicity, pathogenesis, and experimental approaches aimed at limiting disease manifestations. Although the most prominent alphavirus outbreaks in the past 15 years featured chikungunya virus, and a large body of work has been dedicated to understanding chikungunya disease mechanisms, this Review will instead focus on other arthritogenic alphaviruses that have been identified globally and provide a comprehensive appraisal of present and future research directions.

CXCL10 Signaling Contributes to the Pathogenesis of Arthritogenic Alphaviruses.

Emerging and re-emerging arthritogenic alphaviruses, such as Chikungunya virus (CHIKV) and O'nyong nyong virus, cause acute and chronic crippling arthralgia associated with inflammatory immune responses. Approximately 50% of CHIKV-infected patients suffer from rheumatic manifestations that last 6 months to years. However, the physiological functions of individual immune signaling pathways in the pathogenesis of alphaviral arthritis remain poorly understood. Here, we report that a deficiency in CXCL10, which is a chemoattractant for monocytes/macrophages/T cells, led to the same viremia as wild-type animals, but fewer immune infiltrates and lower viral loads in footpads at the peak of arthritic disease (6-8 days post infection). Macrophages constituted the largest immune cell population in footpads following infection, and were significantly reduced in Cxcl10-/- mice. The viral RNA loads in neutrophils and macrophages were reduced in Cxcl10-/- compared to wild-type mice. In summary, our results demonstrate that CXCL10 signaling promotes the pathogenesis of alphaviral disease and suggest that CXCL10 may be a therapeutic target for mitigating alphaviral arthritis.

Endemic and Epidemic Human Alphavirus Infections in Eastern Panama: An Analysis of Population-Based Cross-Sectional Surveys.

Madariaga virus (MADV) has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June 2017, a fatal MADV infection was confirmed in a community of Darien Province. We conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. In addition, by applying a catalytic, force-of-infection (FOI) statistical model to two serosurveys from Darien Province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. In 2017, MADV and VEEV IgM seroprevalences were 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%, VEEV: 16.8%, Una virus (UNAV): 16.0%, and Mayaro virus: 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households, raising questions about its vectors and enzootic transmission cycles. Insomnia was associated with MADV and VEEV infections, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in Aruza and Mercadeo, respectively. The lack of additional neurological cases suggests that severe MADV and VEEV infections occur only rarely. Our results indicate that over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased-potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially in some locations of eastern Panama.

Infectious cDNA clones of two strains of Mayaro virus for studies on viral pathogenesis and vaccine development.

Mayaro virus (MAYV; family Togaviridae, genus Alphavirus) is an emerging global threat that can cause severe clinical manifestations similar to Zika, dengue, and chikungunya viruses. Currently, there is a lack of molecular tools to enable a better understanding of the transmission and pathogenesis of MAYV. Here, we detail the development and characterization of infectious clones of two strains of MAYV that produce infectious virus and replicate in mammalian and mosquito cells similarly to wild-type virus. Additionally, clone-derived viruses produced identical infection rates and phenotypes in CD-1 mice compared to the parental strains. This infectious clone system will provide a resource to the research community to analyze MAYV genetic determinants of virulence, determine vector competence, and develop vaccines.

Pancreas disease caused by Salmonid alphavirus subtype 2 reduces growth and feed conversion in farmed Atlantic salmon.

Pancreas disease (PD), caused by several subtypes of salmonid alphavirus (SAV), is associated with significant economic losses in European salmonid aquaculture. In this retrospective cohort study, we investigate the impact of PD caused by SAV subtype 2 (SAV2) on growth, feed conversion, and mortality in farmed Atlantic salmon (Salmo salar L.). The study was based on harvest data from a large salmon farming company operating in the SAV2 endemic area of Norway. Mixed-effect regression analyses showed a severe impact on both growth and feed conversion when PD appeared late in the production cycle. In a scenario with fixed slaughter time the estimated impact corresponded to a growth reduction of 0.7 kg and 0.07 points increase in feed conversion ratio. No effect on mortality was observed in this data set. In conclusion, the most important consequences of PD caused by SAV2 infection is reduced growth and feed conversion in large Atlantic salmon. The lack of effect on mortality in this study may be due to other factors overshadowing the impact of PD.

Inhibition of Interleukin-1ß Signaling by Anakinra Demonstrates a Critical Role of Bone Loss in Experimental Arthritogenic Alphavirus Infections.

OBJECTIVE: Arthritogenic alphaviruses, such as Ross River virus (RRV) and chikungunya virus (CHIKV), particularly affect joints of the extremities and can lead to debilitating and potentially chronic polyarthritis/polyarthralgia. The innate immune response of the host plays a crucial role in inducing proinflammatory host factors, leading to tissue destruction and bone loss in the joints. This study was performed to assess how the inhibition of interleukin-1ß (IL-1ß) signaling using the clinical rheumatoid arthritis drug anakinra influences bone loss in mice with arthritogenic alphavirus infections. METHODS: Mice (n = 5 per group) were infected with RRV or CHIKV and then treated with anakinra. Weight gain and disease severity were measured, tissue viral titers were determined, and histologic changes in joint tissues were assessed. RESULTS: Anakinra therapy reduced RRV- and CHIKV-induced bone loss in this murine model (P < 0.001 and P < 0.05, respectively). Histologic analysis of the knee joint showed that treatment with anakinra decreased epiphyseal growth plate thinning, loss of epiphyseal bone volume, and osteoclastogenesis in the tibia. Importantly, pharmacologic IL-1 receptor (IL-1R) blockade did not improve other clinical features, including disease score, weight loss, or viremia. CONCLUSION: The present findings suggest that anakinra therapy may reduce bone loss in experimental murine models of RRV and CHIKV. Further investigations are needed to assess the potential therapeutic benefits of anakinra in patients with arthritogenic alphavirus disease.

Interferon regulatory factors 3 and 7 have distinct roles in the pathogenesis of alphavirus encephalomyelitis.

Interferon (IFN) regulatory factors (IRFs) are important determinants of the innate response to infection. We evaluated the role(s) of combined and individual IRF deficiencies in the outcome of infection of C57BL/6 mice with Sindbis virus, an alphavirus that infects neurons and causes encephalomyelitis. The brain and spinal cord levels of Irf7, but not Irf3 mRNAs, were increased after infection. IRF3/5/7-/- and IRF3/7-/- mice died within 3-4 days with uncontrolled virus replication, similar to IFNα receptor-deficient mice, while all wild-type (WT) mice recovered. IRF3-/- and IRF7-/- mice had brain levels of IFNα that were lower, but brain and spinal cord levels of IFNß and IFN-stimulated gene mRNAs that were similar to or higher than WT mice without detectable serum IFN or increases in Ifna or Ifnb mRNAs in the lymph nodes, indicating that the differences in outcome were not due to deficiencies in the central nervous system (CNS) type I IFN response. IRF3-/- mice developed persistent neurological deficits and had more spinal cord inflammation and higher CNS levels of Il1b and Ifnγ mRNAs than WT mice, but all mice survived. IRF7-/- mice died 5-8 days after infection with rapidly progressive paralysis and differed from both WT and IRF3-/- mice in the induction of higher CNS levels of IFNß, tumour necrosis factor (TNF) α and Cxcl13 mRNA, delayed virus clearance and more extensive cell death. Therefore, fatal disease in IRF7-/- mice is likely due to immune-mediated neurotoxicity associated with failure to regulate the production of inflammatory cytokines such as TNFα in the CNS.

High costs of infection: Alphavirus infection reduces digestive function and bone and feather growth in nestling house sparrows (Passer domesticus).

Increasingly, ecoimmunology studies aim to use relevant pathogen exposure to examine the impacts of infection on physiological processes in wild animals. Alphaviruses are arthropod-borne, single-stranded RNA (ssRNA) viruses ("arboviruses") responsible for millions of cases of human illnesses each year. Buggy Creek virus (BCRV) is a unique alphavirus that is transmitted by a cimicid insect, the swallow bug, and is amplified in two avian species: the house sparrow (Passer domesticus) and the cliff swallow (Petrochelidon pyrrhonota). BCRV, like many alphaviruses, exhibits age-dependent susceptibility where the young are most susceptible to developing disease and exhibit a high mortality rate. However, alphavirus disease etiology in nestling birds is unknown. In this study, we infected nestling house sparrows with Buggy Creek virus and measured virological, pathological, growth, and digestive parameters following infection. Buggy Creek virus caused severe encephalitis in all infected nestlings, and the peak viral concentration in brain tissue was over 34 times greater than any other tissue. Growth, tissue development, and digestive function were all significantly impaired during BCRV infection. However, based on histopathological analysis performed, this impairment does not appear to be the result of direct tissue damage by the virus, but likely caused by encephalitis and neuronal invasion and impairment of the central nervous system. This is the first study to examine the course of alphavirus diseases in nestling birds and these results will improve our understanding of age-dependent infections of alphaviruses in vertebrate hosts.

Tropical arthritogenic alphaviruses. / Alfavirus tropicales artritogénicos.

Tropical alphaviruses have special tropism for bone and joint tissue. Patients can develop chronic rheumatic disorders similar to rheumatoid arthritis and ankylosing spondylitis. The prototype is Chikungunya virus, although other lesser known viruses in our environment such as Sindbis, Ross River, Mayaro, O'nyong nyong and Barmah Forest viruses have the potential to be sped through vectors and cause chronic rheumatic disease. International population movements have increased the numbers of patients diagnosed with these tropical viruses in areas in which they are not endemic. Since they can leave persistent symptoms and affect the quality of life of the patients, it is important that we be aware of them. Changes in ecosystems have favored the expansion of competent mosquitoes, making fears of local transmission in southern Europe a reality. The objective of this review is to provide a clinical approach to the different arthritogenic tropical alphaviruses, especially those in which chronic rheumatic disease is more frequent.

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus.

UNLABELLED: The alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

Kinetics of chikungunya infections during an outbreak in Southern Thailand, 2008-2009.

The Indian Ocean chikungunya epidemic re-emerged in Thailand in August 2008. Forty-five adults with laboratory-confirmed chikungunya in Songkhla province, Thailand were clinically assessed and serially bled throughout the acute and convalescent phase of the disease. Patient symptoms, antibody responses, and viral kinetics were evaluated using observational assessments, polymerase chain reaction (PCR), and serological assays. All subjects experienced joint pain with 42 (93%) involving multiple joints; the interphalangeal most commonly affected in 91% of the subjects. The mean duration of joint pain was 5.8 days, 11 (25%) experiencing discomfort through the duration of the study. Rash was observed in 37 (82%) subjects a mean 3.5 days post onset of symptoms. Patents were positive by PCR for a mean of 5.9 days with sustained peak viral load through Day 5. The IgM antibodies appeared on Day 4 and peaked at Day 7 and IgG antibodies first appeared at Day 5 and rose steadily through Day 24.

Cross-inhibition of chikungunya virus fusion and infection by alphavirus E1 domain III proteins.

Alphaviruses are small enveloped RNA viruses that include important emerging human pathogens, such as chikungunya virus (CHIKV). These viruses infect cells via a low-pH-triggered membrane fusion reaction, making this step a potential target for antiviral therapies. The E1 fusion protein inserts into the target membrane, trimerizes, and refolds to a hairpin-like conformation in which the combination of E1 domain III (DIII) and the stem region (DIII-stem) pack against a core trimer composed of E1 domains I and II (DI/II). Addition of exogenous DIII proteins from Semliki Forest virus (SFV) has been shown to inhibit E1 hairpin formation and SFV fusion and infection. Here we produced and characterized DIII and DI/II proteins from CHIKV and SFV. Unlike SFV DIII, both core trimer binding and fusion inhibition by CHIKV DIII required the stem region. CHIKV DIII-stem and SFV DIII-stem showed efficient cross-inhibition of SFV, Sindbis virus, and CHIKV infections. We developed a fluorescence anisotropy-based assay for the binding of SFV DIII-stem to the core trimer and used it to demonstrate the relatively high affinity of this interaction (Kd [dissociation constant], ∼85 nM) and the importance of the stem region. Together, our results support the conserved nature of the key contacts of DIII-stem in the alphavirus E1 homotrimer and describe a sensitive and quantitative in vitro assay for this step in fusion protein refolding.

Deciphering the host-pathogen protein interface in chikungunya virus-mediated sickness.

Successful infection with chikungunya virus (CHIKV) depends largely on the ability of this virus to manipulate cellular processes in its favour through specific interactions with several host factors. The knowledge of virus-host interactions is of particular value for understanding the interface through which therapeutic strategies could be applied. In the current study, the authors have employed a computational method to study the protein interactions between CHIKV and both its human host and its mosquito vector. In this structure-based study, 2028 human and 86 mosquito proteins were predicted to interact with those of CHIKV through 3918 and 112 unique interactions, respectively. This approach could predict 40 % of the experimentally confirmed CHIKV-host interactions along with several novel interactions, suggesting the involvement of CHIKV in intracellular cell signaling, programmed cell death, and transcriptional and translational regulation. The data corresponded to those obtained in earlier studies for HIV and dengue viruses using the same methodology. This study provides a conservative set of potential interactions that can be employed for future experimental studies with a view to understanding CHIKV biology.

Underdiagnosis of chikungunya virus infections in symptomatic dutch travelers returning from the Indian ocean area.

A putative underdiagnosis of clinical chikungunya virus infection in Dutch travelers to the Indian Ocean area was addressed by retrospective screening of all sera for which requested dengue virus serology was negative in the period 2007 to 2010. Evidence for a recent infection was observed in 6.5% of 107 patients, indicating a substantial underdiagnosis and the need for increased awareness among physicians.

Morbidity and impaired quality of life 30 months after chikungunya infection: comparative cohort of infected and uninfected French military policemen in Reunion Island.

We compared the morbidity and quality of life of military policemen ("gendarmes") infected with chikungunya virus (CHIKV+) 30 months after contamination. We categorized the subjects in 3 groups: healed patients (n = 48), non-healed patients (n = 37, 44% of CHIKV+), and uninfected subjects (CHIKV-, n = 297). Data were self-recorded in this retrospective cohort study; they included sociodemographic information, clinical symptoms, and the Medical Outcome Study 36-item short-form health survey (MOS-SF36) quality of life questionnaire. The study population was mostly men (92%), with a median age of 42.8 years, regardless of CHIKV status. The main complaints were rheumatic symptoms (pain, stiffness, and swelling), reported 5 times more often by non-healed CHIKV+ subjects and 2-3 times more often by healed CHIKV+ subjects than by CHIKV- subjects, and fatigue. The CHIKV+ patients reported more use of health care services. Thirty months after infection, all rheumatic symptoms were more frequent and intense among CHIKV+ than among CHIKV- subjects, with a gradient of severity between healed and non-healed CHIKV+ subjects. Non-healed CHIKV+ subjects reported subsequent limitation in their activities. All dimensions of MOS-SF36 as well as physical and mental component summaries were impaired in CHIKV+ compared to CHIKV- subjects, with a decreasing gradient of impairment from non-healed to healed CHIKV+ subjects, then to CHIKV- subjects. These observations confirm the long-term impact of CHIKV infection on both physical and mental health. Questions persist regarding the duration of this impairment and the possibility of a return to "before CHIKV" health status for infected patients.

Chikungunya virus-induced autophagy delays caspase-dependent cell death.

Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α-XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16L(HM) mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease.

Accumulation of autophagosomes in Semliki Forest virus-infected cells is dependent on expression of the viral glycoproteins.

Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH(4)Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH(4)Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.

Differential diagnosis of Chikungunya, dengue viral infection and other acute febrile illnesses in children.

BACKGROUND: Clinical manifestations of chikungunya (CHIK) are similar to those of dengue. It would be useful to be able to identify clinical manifestations that could reliably help to differentiate CHIK from dengue and other acute febrile illnesses during a CHIK outbreak in a dengue-endemic area. METHODS: A prospective cohort study was conducted between April and July 2009 in children aged 1 month to 15 years who lived in a CHIK outbreak area in southern Thailand and who had fever <7 days with arthralgia/arthritis, myalgia or rash. CHIK was confirmed by real-time polymerase chain reaction or the indirect immunofluorescence test. RESULTS: Fifty patients were suspected of having CHIK, of whom 32 were confirmed, 1 had coinfection with dengue viral infection (DVI), 10 had dengue alone and 7 had an acute febrile illness. The specificity and positive predictive value of fever and arthralgia together to diagnose CHIK were 47.1% and 74.2%, and the corresponding values of the standard clinical triad (fever, arthralgia, rash) were 70.6% and 83.3%, respectively. Fever ≤ 2 days, skin rash during fever and white blood cell count ≥ 5000 cells/mm(3) were independently and significantly associated with CHIK in comparison with DVI and acute febrile illnesses, with relative risk ratios (95% confidence intervals) of 10.4 (0.9-116) and 13.7 (1.3-145), 13.8 (1.2-164) and 14.8 (1.6-168), and 18.3 (1.7-194) and 1.8 (0.1-20.6), respectively. CONCLUSIONS: During a CHIK outbreak in a DVI-endemic area, overdiagnosis of CHIK was common. Skin rash during fever and white blood cell count ≥ 5000 cells/mm(3) or specific antigen testing (if available) can be helpful in differentiating CHIK from DVI.

Genetic associations of fatigue and other symptom domains of the acute sickness response to infection.

The acute sickness response to infection is a conserved set of changes in physiology and behaviour, featuring fever, fatigue, musculo-skeletal pain, disturbed mood, and cognitive difficulties. The manifestations differ somewhat between individuals, including those infected with pathogens which do not have genetic variability--suggesting host determinants. Principal components analysis (PCA) was applied to acute phase, self-report symptom data from subjects in the Dubbo Infection Outcomes Study (n=296) to empirically derive indices of fatigue, pain, neurocognitive difficulties, and mood disturbance, as well as overall illness severity. Associations were sought with functional single nucleotide polymorphisms (SNPs) in the cytokine genes, interleukin (IL)-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-10. The summed individual symptom indices correlated with overall severity and also with functional status. The relative contribution of individual symptom domains to the overall illness was stable over time within subjects, but varied between subjects with the same infection. The T allele of the IFN-γ +874 T/A SNP was associated with increased fatigue (p=0.0003; OR: 3.3). The C allele of the IL-10 -592 C/A SNP exerted a protective effect on neurocognitive difficulties (p=0.017; OR: 0.52); while the A allele for the IL-10 -592 SNP was associated with increased mood disturbance (p=0.044; OR: 1.83), as was the G allele of the IL-6 -174 G/C SNP (p=0.051; OR: 1.83). The acute sickness response has discrete symptom domains including fatigue, which have unique genetic associations. These data provide novel insights into the pathophysiology of fatigue states.